The invention relates generally to plant galactolipids and more particularly the gene encoding digalactosyldiacylglycerol galactosyltransferase.
The process of photosynthesis is the basis for all life on earth because it provides oxygen and ultimately converts inorganic matter into organic matter. The photosynthetic apparatus in plant cells is associated with a particular membrane system inside chloroplasts, the thylakoids. Four lipids are found to be associated with thylakoid membranes in plants and photosynthetic bacteria. Only one of them is a phospholipid, the ubiquitous phosphatidylglycerol. The other three are non-phosphorous diacylglycerol glycolipids with one or two galactose moieties or a sulfonic acid derivative of glucose attached to diacylglycerol. Browse, J. et al., Ann. Rev. Plant Physiol. Plant Mol. Biol. 42:467 (1991); Joyard, J. et al., Plant Physiol. 118:715 (1998). The galactolipids constitute the bulk (close to 80%) of the thylakoid lipid matrix and within green plant parts, 70-80% of the lipids are associated with photosynthetic membranes. Taking into account that plants represent the major portion of the global bioorganic matter, it comes as no surprise that the two galactolipids, mono- and digalactosyldiacylglycerol, are the most abundant lipids in the biosphere. Most vegetables and fruits in human or animal diets are rich in galactolipids. Their breakdown products represent an important dietary source of galactose and polyunsaturated fatty acids. Ohlsson, L. et al., J. Nutrition 128:239 (1998); Andersson, L. et al., J. Lipid Res. 36:1392 (1995). The elucidation of the pathway for galactolipid biosynthesis has been extremely challenging. Thylakoid membrane lipid biosynthesis in plants is highly complex bringing together carbohydrate and fatty acid metabolisms. There is a mesmerising number of molecular species for each thylakoid lipid due to the large number of combinatorial possibilities for fatty acid substituents. Even more dazzling, the biosynthesis of thylakoid lipids is not restricted to enzymes associated with the chloroplast where galactolipids are found, but also involves enzymes in the endoplasmic reticulum (ER) (FIG. 1). The mechanism for subcellular trafficking of lipid moieties from the ER that ultimately become incorporated into the thylakoid lipids inside the plastids poses one of the most challenging enigmas of modern plant biochemistry. Molecular species of galactolipids containing diacylglycerol moieties derived from the plastid or the ER pathway can be distinguished based on their fatty acid composition. Heinz, E. et al., Plant Physiol. 72:273 (1983). Lipid moieties assembled inside the plastid carry preferentially a 16-carbon fatty acid in the sn2-position of diacylglycerol, while lipids derived from the ER pathway contain an 18-carbon fatty acid in this position. This is due to different substrate specificities of the respective acyltransferases in the plastid and the ER. An extensive screening of different plant species revealed that the plastid pathway is dispensable in many plants. Mongrand, S. et al., Phytochemistry 49:1049 (1998). However, no naturally occurring plant has been found, in which the ER pathway was non-functional. A mutant of Arabidopsis, act1, is partially blocked in the plastid pathway. Kunst, L. et al., PNAS (USA) 85:4143 (1988). This mutant is deficient in the acyltransferase which catalyses the biosynthesis of lysophosphatidic acid inside the plastid (FIG. 1). Other mutants of Arabidopsis have been described that affect the fatty acid and, thus, the molecular species composition of thylakoid lipids. Browse, J. et al., in Arabidopsis, E. M. Meyerowitz and C. R. Somerville, Eds. (Cold Spring Harbor Laboratory Press, N.Y.) pp. 881-912 (1994). Most of these are deficient in fatty acid desaturases. However, the only higher plant mutant known to be directly affected in galactolipid assembly is the dgd1 mutant of Arabidopsis. Dormann, P. et al., Plant Cell 7:1801 (1995). In this mutant the relative amount of the digalactosyl lipid is reduced to 10% of wild type. It has already proven to be very valuable in assessing the importance of the digalactosyl lipid for the assembly and function of the photosynthetic membranes. Growth, chloroplast ultra structure, the composition and relative ratios of different pigment protein complexes, the light utilization by the photosynthetic apparatus, and the import of proteins into chloroplasts are affected in the dgd1 mutant. Hartel, H. et al., Plant Physiol. 115:1175 (1997); Reifarth, F. et al., Biochemistry 36:11769 (1997); Hartel, H. et al., Plant Physiol. Biochem. 36:407 (1998); Chen, L.-J. et al., Plant J. 16:33 (1998). In addition to the reduction in the amount of galactolipid, the dgd1 mutant also shows a peculiar alteration in the fatty acid composition of the monogalactosyl lipid with a characteristic increase in the amount of molecular species containing 18-carbon fatty acids. The accumulation of these molecular species of the monogalactosyl lipid is consistent with their presumed precursor function in the biosynthesis of the digalactosyl lipid. Based on labelling experiments with isolated chloroplasts (van Besouw, A. et al., Biochim. Biophys. Acta 529:44 (1978); Hemmskerk, J. W. M. et al., Plant Physiol. 93:1286 (1990)), it has been proposed that one galactose moiety is transferred from one monogalactosyl lipid onto a second to form the digalactosyl lipid (FIG. 1). The released diacylglycerol moiety is made available for further thylakoid lipid assembly with the bulk appearing in monogalactosyl lipid. As can be assumed from the fatty acid composition of the digalactosyl lipid in the wild type (Browse, J. et al., Biochem. J. 235:25 (1986)), the responsible enzyme is specific for molecular species derived from the ER. Accordingly, approximately equal amounts of ER-derived molecular species are found in the digalactosyl and monogalactosyl lipids (FIG. 1). Therefore, it is expected that the disruption of digalactosyl lipid biosynthesis in the dgd1mutant also disturbs the assembly of other thylakoid lipids, in particular the ER-derived monogalactosyl lipid.
It would thus be desirable to provide the wild-type DGD1 gene encoding for digalactosyldiacylglycerol galactosyltransferase (DGD1). It would also be desirable to isolate and purify the gene product. It would be further desirable to provide in vitro and in vivo assays to screen for new herbicides that inhibit the DGD1 gene product. Galactolipids are unique to plants and other photosynthetic organisms. Therefore, in contrast to most herbicides currently in use, herbicides that inhibit galactolipid biosynthesis will not be toxic to animals, humans or microbial organisms in the soil.
It would also be desirable to control the digalactosyldiacylglycerol levels in plants by controlling the expression of the gene encoding for the DGD1 protein. It would further be desirable to transform plants using the gene in order to alter their lipid composition. An alteration in lipid composition would provide plants with an increased resistance to environmental factors such as, but not limited to, temperature stress and/or pathogen infection. It would further provide an increase in the yield of crop plants such as leafy vegetables.
The present invention provides a novel purified and isolated nucleic acid sequence encoding digalactosyldiacylglycerol galactosyltransferase (DGD1). The cDNA encoding DGD1 is set forth SEQ ID NO: 1. The deduced amino acid sequence of DGD1 is also provided and set forth in SEQ ID NO: 2. The protein has a predicted molecular weight of 91.8 kDa and has some sequence similarity in the C-terminal portion to bacterial and plant glycosyltransferases.
Methods for making and using the cDNA encoding DGD1 are also provided. For example, wild-type DGD1 can be used to produce recombinant DGD1 in bacteria or yeast. Such recombinant protein can be used in either an in vivo or in vitro assay to screen compounds for new herbicides. Additionally, DGD1 may be used to alter a plant""s leaf lipid composition thus altering sensitivity to environmental factors such as, but not limited to, temperature stress and/or pathogen infection and, in some cases, increase the yield of crop plants. Expression vectors containing the cDNA, transgenic plants and other organisms, e.g., E. coli, transfected with said vectors, as well as seeds from said plants, are also provided by the present invention.